Ring-opening cross-metathesis (ROCM) as a novel tool for the ligation of peptides

The development of ring-opening cross-metathesis (ROCM) as a novel tool for the site-specific ligation of peptide units is reported. The resulting structural units at the site of ligation resulting from ROCM resemble proline as well as other known beta-turn stabilising structural units. ROCM under mild reaction conditions between a variety of peptides bearing a cyclic olefin with amino acids or peptides results in high yields. The peptidic cross-partners for metathesis are equipped with double bonds via the N and the C terminus and the side chain, respectively, to allow the synthesis of linear as well as non-linear and branched peptides. The ligation in this manner succeeds with low catalyst loadings, with no need for any excess of one reaction partner and with a high compatibility with a wide range of functional groups. Furthermore, the stereochemical outcome of the ROCM can easily be controlled by using a Hoveyda-type chiral catalyst. Fluorescence labelling of peptides is possible in the same manner when using a cyclic olefin equipped with a fluorescence marker.     

Extending synthetic access to proteins with a removable acyl transfer auxiliary

A chemo- and regioselective auxiliary-mediated peptide ligation has been developed that is effective under nonidealized conditions for the synthesis of proteins. This general amide bond ligation utilizes a removable auxiliary that is analogous to the role of cysteine in native chemical ligation, combining chemoselective thioester exchange with efficient regioselective intramolecular acyl transfer. Acid lability and improved ligation efficiency were introduced into the 2-mercaptobenzyl auxiliary by increasing the electron density of the aromatic ring. The 62 amino acid SH3 domain from alpha-spectrin was synthesized using the auxiliary-mediated ligation at a Lys-Gly sequence. The auxiliary was removed with TFA and scavengers from the ligated product. This methodology enables unprotected peptides to be coupled at noncysteine ligation sites expanding the scope of protein synthesis and semisynthesis.     

Efficient cleavage of the N-O bond of 3,6-dihydro-1,2-oxazines mediated by some alpha-hetero substituted carbonyl compounds in mild conditions

The efficient cleavage of the N-O bond of some nitroso Diels-Alder cycloadducts has been achieved in mild conditions, mediated either by 2,2-dimethyl-1,3-dioxan-5-one or 1,3-dithiolane-2-carboxaldehyde. These new and purely organic conditions allow an excellent tolerance with respect to many functional groups that would have been affected by previous reductive cleavage conditions.     

Regio- and chemoselective covalent immobilization of proteins through unnatural amino acids

A general approach was developed for the regio- and chemoselective covalent immobilization of soluble proteins on glass surfaces through an unnatural amino acid created by post-translationally modifying the cysteine residue in a CaaX recognition motif with functional groups suitable for "click" chemistry or a Staudinger ligation. Farnesyl diphosphate analogues bearing omega-azide or omega-alkyne moieties were attached to the cysteine residue in Cys-Val-Ile-Ala motifs at the C-termini of engineered versions of green fluorescent protein (GFP) and glutathione S-transferase (GST) by protein farnesyltransferase. The derivatized proteins were attached to glass slides bearing linkers containing azide ("click" chemistry) or phosphine (Staudinger ligation) groups. "Click"-immobilized proteins were detected by fluorescently labeled antibodies and remained attached to the slide through two cycles of stripping under stringent conditions at 80 degrees C. GFP immobilized by a Staudinger ligation was detected by directly imagining the GFP fluorophore over a period of 6 days. These methods for covalent immobilization of proteins should be generally applicable. CaaX recognition motifs can easily be appended to the C-terminus of a cloned protein by a simple modification of the corresponding gene, and virtually any soluble protein or peptide bearing a CaaX motif is a substrate for protein farnesyltransferase.     

Site-specific protein immobilization by Staudinger ligation

The Staudinger ligation between an azido-protein and a phosphinothioester-derivatized surface is demonstrated to be an effective means for the site-specific, covalent immobilization of a protein. Immobilization yields of >50% are obtained in <1 min, and immobilized proteins have >80% of their expected activity. No other method enables more rapid immobilization or a higher yield of active protein. Because azido-peptides and azido-proteins are readily attainable by synthesis, biosynthesis, or semisynthesis, the Staudinger ligation could be of unsurpassed utility in creating microarrays of functional peptides and proteins.     

Peptide/protein-polymer conjugates: synthetic strategies and design concepts

This feature article provides a compilation of tools available for preparing well-defined peptide/protein-polymer conjugates, which are defined as hybrid constructs combining (i) a defined number of peptide/protein segments with uniform chain lengths and defined monomer sequences (primary structure) with (ii) a defined number of synthetic polymer chains. The first section describes methods for post-translational, or direct, introduction of chemoselective handles onto natural or synthetic peptides/proteins. Addressed topics include the residue- and/or site-specific modification of peptides/proteins at Arg, Asp, Cys, Gln, Glu, Gly, His, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val residues and methods for producing peptides/proteins containing non-canonical amino acids by peptide synthesis and protein engineering. In the second section, methods for introducing chemoselective groups onto the side-chain or chain-end of synthetic polymers produced by radical, anionic, cationic, metathesis and ring-opening polymerization are described. The final section discusses convergent and divergent strategies for covalently assembling polymers and peptides/proteins. An overview of the use of chemoselective reactions such as Heck, Sonogashira and Suzuki coupling, Diels-Alder cycloaddition, Click chemistry, Staudinger ligation, Michael's addition, reductive alkylation and oxime/hydrazone chemistry for the convergent synthesis of peptide/protein-polymer conjugates is given. Divergent approaches for preparing peptide/protein-polymer conjugates which are discussed include peptide synthesis from synthetic polymer supports, polymerization from peptide/protein macroinitiators or chain transfer agents and the polymerization of peptide side-chain monomers.     

Per(poly)fluoroalkanesulfinamides assisted diastereoselective three-component inverse-electron-demand aza Diels-Alder reaction

A highly diastereoselective three-component inverse-electron-demand aza Diels-Alder reaction assisted by per(poly)fluoro-alkanesulfinamides is presented, providing a broad spectrum of highly functionalized piperidine derivatives with excellent endo/exo and facial diastereoselectivities. The electron-withdrawing perfluoroalkyl groups are crucial for the success of this reaction under mild conditions and facilitate monitoring the process and stereoselectivities of the reaction. The synthetic potential of these cycloadducts is also highlighted.     

Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization.

The highly chemoselective reaction between unprotected peptides bearing an N-terminal Cys residue and a C-terminal thioester enables the total and semi-synthesis of complex polypeptides. Here we extend the utility of this native chemical ligation approach to non-cysteine containing peptides. Since alanine is a common amino acid in proteins, ligation at this residue would be of great utility. To achieve this goal, a specific alanine residue in the parent protein is replaced with cysteine to facilitate synthesis by native chemical ligation. Following ligation, selective desulfurization of the resulting unprotected polypeptide product with H(2)/metal reagents converts the cysteine residue to alanine. This approach, which provides a general method to prepare alanyl proteins from their cysteinyl forms, can be used to chemically synthesize a variety of polypeptides, as demonstrated by the total chemical syntheses of the cyclic antibiotic microcin J25, the 56-amino acid streptococcal protein G B1 domain, and a variant of the 110-amino acid ribonuclease, barnase.     

Hetero Diels-Alder reactions of 1-amino-3-siloxy-1,3-butadienes under strictly thermal conditions

The hetero Diels-Alder reaction of 1-amino-3-siloxy-1,3-butadiene (1a) with a range of unactivated aldehydes proceeds readily under remarkably mild conditions: at room temperature and in the absence of Lewis acid catalysts. The cycloadducts are formed in good yields and can be converted directly to the corresponding dihydro-4-pyrones using acetyl chloride. Ketones and imines are also reactive in hetero Diels-Alder reactions with this diene.     

2,4-Bis(methylsulfanyl)pyrimidine o-quinodimethane: a versatile building block for functionalized fused pyrimidines

Two alternative methods for the preparation of new pyrimidine Diels-Alder cycloadducts from the readily available 2,4-bis(methylsulfanyl)-5,6-dihydrocyclobuta[d]pyrimidine are presented. In the first method, the in situ generated pyrimidine ortho-quinodimethane reacts with various dienophiles to form the respective cycloadducts bearing two methylthio groups, which can be easily replaced by other functional groups. In the second method, one or both of the methylsulfanyl groups of the starting pyrimidine are replaced first and the resulting functionalized pyrimidines are able to undergo Diels-Alder cyclization with different dienophiles to form pyrimidine cycloadducts. These alternative synthetic strategies provide access to a wide variety of pyrimidine cycloadducts with a different substitution pattern on the pyrimidine ring. Yield data indicate that the electronic nature of the functional groups strongly influence the efficiency of the cycloaddition reaction.     

Fmoc-based synthesis of peptide alpha-thioesters using an aryl hydrazine support

C-Terminal peptide thioesters are key intermediates in the synthesis/semisynthesis of proteins and of cyclic peptides by native chemical ligation. They are prepared by solid-phase peptide synthesis (SPPS) or biosynthetically by protein splicing techniques. Until recently, the chemical synthesis of C-terminal alpha-thioester peptides by SPPS was largely restricted to the use of Boc/Benzyl chemistry due to the poor stability of the thioester bond to the basic conditions required for the deprotection of the N(alpha)-Fmoc group. In the present work, we describe a new method for the SPPS of C-terminal thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. This step converts the acyl hydrazine group into a highly reactive acyl diazene intermediate which reacts with an alpha-amino acid alkyl thioester (H-AA-SR) to yield the corresponding peptide alpha-thioester in good yield. This method has been successfully used to prepare a variety of peptide thioesters, cyclic peptides, and a fully functional Src homology 3 (SH3) protein domain.     

The reaction of selenoaldehydes with 2-methoxyfuran using their generation by retro Diels-Alder reaction

Selenoaldehydes, regenerated by thermal retro Diels-Alder reaction of anthracene cycloadducts under neutral conditions, reacted with 2-methoxyfuran to give methyl penta-2,4-dienoates along with the deposition of elemental selenium. In a similar reaction with 2-methoxyfuran using thioaldehyde, thiirane compound was isolated. This suggests the formation of selenirane intermediates in the above reaction.     

Mass spectrometric identification of formaldehyde-induced peptide modifications under in vivo protein cross-linking conditions

Formaldehyde cross-linking of proteins is emerging as a novel approach to study protein-protein interactions in living cells. It has been shown to be compatible with standard techniques used in functional proteomics such as affinity-based protein enrichment, enzymatic digestion, and mass spectrometric protein identification. So far, the lack of knowledge on formaldehyde-induced protein modifications and suitable mass spectrometric methods for their targeted detection has impeded the identification of the different types of cross-linked peptides in these samples. In particular, it has remained unclear whether in vitro studies that identified a multitude of amino acid residues reacting with formaldehyde over the course of several days are suitable substitutes for the much shorter reaction times of 10-20 min used in cross-linking experiments in living cells. The current study on model peptides identifies amino-termini as well as lysine, tryptophan, and cysteine side chains, i.e. a small subset of those modified after several days, as the major reactive sites under such conditions, and suggests relative position in the peptide sequence as well as sequence microenvironment to be important factors that govern reactivity. Using MALDI-MS, mass increases of 12 Da on amino groups and 30 Da on cysteines were detected as the major reaction products, while peptide fragment ion analysis by tandem mass spectrometry was used to localize the actual modification sites on a peptide. Non-specific cross-linking was absent, and could only be detected with low yield at elevated peptide concentrations. The detailed knowledge on the constraints and products of the formaldehyde reaction with peptides after short incubation times presented in this study is expected to facilitate the targeted mass spectrometric analysis of proteins after in vivo formaldehyde cross-linking.     

A Disulfide Intercalator Toolbox for the Site‐Directed Modification of Polypeptides

A disulfide intercalator toolbox was developed for site‐specific attachment of a broad variety of functional groups to proteins or peptides under mild, physiological conditions. The peptide hormone somatostatin (SST) served as model compound for intercalation into the available disulfide functionalization schemes starting from the intercalator or the reactive SST precursor before or after bioconjugation. A tetrazole–SST derivative was obtained that undergoes photoinduced cycloaddition in mammalian cells, which was monitored by live‐cell imaging.     

Visible-Light-Induced Cysteine-Specific Bioconjugation: Biocompatible Thiol–Ene Click Chemistry

Bioconjugation methods using visible-light photocatalysis have emerged as powerful synthetic tools for the selective modification of biomolecules under mild reaction conditions. However, the number of photochemical transformations that allow successful protein bioconjugation is still limited because of the need for stringent reaction conditions. Herein, we report that a newly developed water-compatible fluorescent photosensitizer Q_PEG can be used for visible-light-induced cysteine-specific bioconjugation for the installation of Q_PEG by exploiting its intrinsic photosensitizing ability to activate the S−H bond of cysteine. The slightly modified Q_CAT enables the effective photocatalytic cysteine-specific conjugation of biologically relevant groups. The superior reactivity and cysteine selectivity of this methodology was further corroborated by traceless bioconjugation with a series of complex peptides and proteins under biocompatible conditions.     

Selective synthesis of multiply substituted 7-norbornenone derivatives or Diels-Alder cycloadducts from 1,2,3,4-tetrasubstituted 1,3-butadienes and maleic anhydride with or without Lewis acids

Reactions of 1,2,3,4-tetrasubstituted 1,3-butadienes with maleic anhydride and other dienophiles were investigated with or without addition of Lewis acid. When the silylated 1,3-butadienes, such as 1,4-bis(trimethylsilyl)-1,3-butadienes or 1-trimethylsilyl-1,3-butadienes, were treated with maleic anhydride in the presence of 2 equiv of AlCl₃, multi-substituted 7-norbornenones of well-defined exo,exo-disubstituted patterns were produced by an unprecedented and synthetically useful tandem process. Although some tetrasubstituted 1,3-butadienes could react directly with maleic anhydride under relative harsh conditions to afford Diels-Alder cycloadducts, the reactions, in the presence of 1 equiv of AlCl₃, afforded the corresponding cycloadducts in higher yields under mild conditions. These results showed that the size and substitution pattern of substituents on the butadienyl skeleton played a very important role in the reactivity of butadienes as a partner for the Diels-Alder reaction and Lewis acid could promote and/or realize the process of a Diels-Alder reaction.     

Bioconjugation of Peptides by Palladium-Catalyzed C−C Cross-Coupling in Water

The abiotic, regioselective conjugation of peptides and proteins with non-proteinogenic structural elements requires mild and fast coupling reactions which are compatible with an aqueous reaction medium and orthogonal in their reactivity with all other functional groups in the protein. Sonogashira coupling with a palladium–guanidinophosphane catalyst that is prepared in situ (see reaction on the right) complies with these demands.     

Indole-derived arynes and their Diels-Alder reactivity with furans

Arynes derived from any position of the ubiquitous indole nucleus are unknown. We have now provided the first evidence for the formation and trapping of the 4,5-, 5,6-, and 6,7-indolynes. A series of o-dihalo indoles (Cl, Br, F) were synthesized and reacted under metal-halogen exchange conditions to give Diels-Alder cycloadducts in high yield with furan. The use of an excess of tert-butyllithium resulted in the rearrangement of the initially formed cycloadduct; however, employing only a slight excess of n-butyllithium cleanly gave cycloadducts with furan.     

Selenolysine: A New Tool for Traceless Isopeptide Bond Formation

Despite their biological importance, post-translationally modified proteins are notoriously difficult to produce in a homogeneous fashion by using conventional expression systems. Chemical protein synthesis or semisynthesis offers a solution to this problem; however, traditional strategies often rely on sulfur-based chemistry that is incompatible with the presence of any cysteine residues in the target protein. To overcome these limitations, we present the design and synthesis of γ-selenolysine, a selenol-containing form of the commonly modified proteinogenic amino acid, lysine. The utility of γ-selenolysine is demonstrated with the traceless ligation of the small ubiquitin-like modifier protein, SUMO-1, to a peptide segment of human glucokinase. The resulting polypeptide is poised for native chemical ligation and chemoselective deselenization in the presence of unprotected cysteine residues. Selenolysine's straightforward synthesis and incorporation into synthetic peptides marks it as a universal handle for conjugating any ubiquitin-like modifying protein to its target.